PT1400252E - Methods and compositions for healing and repair of articular cartilage - Google Patents

Abstract

Methods and compositions are provided for the treatment of articular cartilage defects and disease involving the combination of the tissue, such as osteochondral grafts, with active growth factor. The active growth factor is preferably a composition containing at least one bone morphogenetic protein and a suitable carrier. The method results in the regeneration of functional repair of articular cartilage tissue.

Description

METHODS AND COMPOSITIONS FOR HEALING AND REPAIRING JOINT CARTILAGE "

FIELD OF THE INVENTION The present invention relates to the field of tissue repair, specifically, to the regeneration of repair of stable and functional joint cartilage. Thus, the present invention may be useful in reconstructive surgery or in other processes of regeneration or repair of articular cartilage.

BACKGROUND OF THE INVENTION Repair of joint cartilage lesions remains a challenge in orthopedics of the present times. Several of the current therapeutic strategies are based on grafting of cartilaginous and osteocartilaginous tissues. The autologous osteocartilagine graft provides the most suitable physiological material. However, the donor tissue is limited and frequently requires surgery at a secondary site to collect tissue from the transplant. Accordingly, despite substantial efforts in this field, there remains a need for an effective method for repairing defects and lesions of the articular cartilage which provides adequate physiological repair without the need to collect autologous tissue from the patient. 1

SUMMARY OF THE INVENTION The present invention provides methods and compositions for regenerating the functional and physiologically suitable repair of tissues for the repair of lesions and defects of the articular cartilage. In particular, the present invention comprises methods for treating patients with joint cartilage lesions or defects. The methods and compositions of the present invention are advantageous in that they use bone morphogenetic proteins (BMPs), which are known to have osteogenic and / or chondrogenic properties and which can be produced by recombinant DNA technology and, consequently, have an available potentially unlimited. The methods and compositions of the present invention are furthermore advantageous in that the regeneration of the functional articular cartilage may be accelerated or be of greater strength and final stability and the tissue formed at the site of the defect or lesion is physiologically suitable. The use of BMP to increase repair of joint cartilage defects and lesions may result in improved methods for treating osteoarthritis by eliminating, delaying or thereby reducing the need for artificial hip replacements and other common interventions. Preclinical evaluations indicate that rhBMP-2 improves the early healing of total thickness defects in the articular cartilage in rabbits. 2

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, there are provided methods and compositions for the treatment of patients suffering from some form of joint cartilage lesions or defects. The injury can be the result of acute stress or injury, such as that resulting from participation in sports activities or accidental occurrences that tear, destroy or otherwise injure the articular cartilage.

The methods and composition are advantageous in repairing or improving articular cartilage defects, particularly total thickness defects in the articular cartilage. Other defects may also be treated by the methods and compositions of the present invention, particularly with a further process in which the defect site is further aggravated in order to reach the underlying subcartilage bone.

In the present invention, active growth factor, such as a BMP, is added to a suitable tissue source. The tissue source may be an osteocartilaginous graft, autologous to the patient or may comprise an allograft or artificially prepared tissue. In a preferred embodiment, the tissue source may consist of chondrocyte cell cultures, such as chondrocytes or stem cell cultures, which have been prepared by ex vivo cell culture methods, with or without additional growth factors. For example, see disclosure of US5226914; US5811094; US5053050; US5486359; US5786217 and US5723331. Disclosures of all such applications are hereby incorporated by reference. The tissue can also be collected by traditional means not based on cell cultures, using techniques, such as mosaicoplasty, in which the cartilage is harvested using commercially available instruments, such as Acufex7 [Smith and Nephew, Inc., Andover MA]; COR System [Innovasive Technologies, Marlborough MA]; or Arthrex7 Osteochondral Autograft Transfer System [Arthrex, Munich, Germany]. The collected tissue may be applied directly in the methods of the present invention or be combined with the tissue-based cell culture systems described above.

The active growth factor used in the present invention is preferably the subclass of the proteins generally known as bone morphogenetic proteins (BMPs), which have been disclosed as having osteogenic, chondrogenic and other growth-like activities and of differentiation. These BMPs include rhBMP-2, rhBMP-3, rhBMP-4 (also referred to as rhBMP-2B), rhBMP-5, rhBMP-6, rhBMP-7 (rhOP-1), rhBMP- 12, rhBMP-13, rhBMP-15, rhBMP-16, rhBMP-17, rhBMP-18, rhGDF-1, rhGDF-3, rhGDF-5, rhGDF-6, rhGDF- rhGDF-10, rhGDF-11, rhGDF-12, rhGDF-14. For example, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed in U.S. Patents 5108922; 5,013,649; 5,117,838; 5,106,448; 5,180,076; and 5141905; BMP-8, disclosed in PCT publication WO91 / 18098; and BMP-9, disclosed in PCT publication WO93 / 00432, bmp-10, disclosed in U.S. Patent 5,637,480; BMP-11, disclosed in U.S. Patent 5,639,638 or BMP-12 or BMP-13, disclosed in U.S. Patent 5,655,882, BMP-15, disclosed in U.S. Pat.

U.S. Patent 5,635,372 and BMP-16, disclosed in co-pending patent application Serial No. 08 / 715,202. Other compositions which may also be useful include Vgr-2 and any of the growth and differentiation factors [GDF], including those described in PCT applications W094 / 15965; W094 / 15949; W095 / 01801; WO95 / 01802; W094 / 21681; W094 / 15966; WO95 / 10539; WO96 / 01845; WO96 / 02559 and others. Also the BIP, disclosed in WO94 / 01557; HP00269, disclosed in JP Publication No. 7-250688; and MP52 disclosed in PCT application WO93 / 16099 may be useful in the present invention. Disclosures of all such applications are hereby incorporated by reference. Also useful in the present invention are heterodimers of the above and their modified proteins or their partial elimination products. These proteins may be used individually or in mixtures of two or more and rhBMP-2 is preferred. BMP can be produced recombinantly or purified from a protein composition. BMP may be homodimeric or may be heterodimeric with other BMPs (eg, a heterodimer composed of a monomer of BMP-2 and BMP-6) or with other members of the TGF-β superfamily, such as activins, inhibins and TGF- βΙ (eg, a heterodimer consisting of a monomer of each of a BMP and a related member of the TGF-β superfamily). Examples of such heterodimeric proteins are described, for example, in PCT Patent Application Publication WO 93/09229, the disclosure of which is hereby incorporated by reference. The amount of the osteogenic protein useful herein is the amount effective to stimulate the increased osteogenic activity of infiltrated progenitor cells and will depend on the size and nature of the defect to be treated as well as the vehicle to be used. Generally, the amount of protein to be delivered is in the range of about 0.05 to about 1.5 mg. 5

In a preferred embodiment, the osteogenic protein is administered in conjunction with an effective amount of a protein that is capable of inducing the formation of tendon or ligament-like tissue. Such proteins include BMP-12, BMP-13 and other members of the BMP-12 subfamily, as well as MP52. These proteins and their use in the regeneration of tendon-like tissue and ligament are disclosed in U.S. application Serial No. 08 / 36,270, filed December 22, 1994, the disclosure of which is hereby incorporated by reference. In another preferred embodiment, a heterodimer is administered in which a monomeric unit is an osteogenic protein, such as BMP-2 and another monomeric subunit is a tendon-inducing protein, such as BMP-12, according to the methods described below , to induce the formation of a functional bond between connective tissue and bone.

APPLICATION OF GROWTH FACTOR Growth factor can be applied to the tissue source as a buffer solution. A preferred buffer solution is a composition comprising, in addition to the active growth factor, about 1.0 to about 10.0% (w / v) glycine, about 0.1 to about 5.0% / v) sugar, preferably sucrose, about 1 to about 20 mM glutamic acid hydrochloride and optionally about 0.01 to about 0.1% nonionic surfactant, such as polysorbate 80. The Preferred solutions are from about 1% to about 20% w / v cellulosic carrier / buffer. If desired, a salt may be added. 6

Other materials which may be suitable for use in the application of the growth factors in the methods and compositions of the present invention include hyaluronic acid, surgical suture or mesh, polyglycolonate, temperature sensitive polymers, demineralized bone, minerals and ceramics such as calcium phosphates , hydroxyapatite, etc., as well as combinations of materials described above. However, in the preferred embodiment of the present invention no vehicle is used. The growth factor of the present invention may be applied directly to the tissue and / or the site in need of tissue repair, in a suitable buffer as described above, or in combination with a suitable carrier. For example, growth factor may be physically applied to the tissue by spraying or submersion or by using a brush or other suitable applicator, such as an injection syringe. Alternatively or in combination, the protein may be applied directly to the site in need of tissue repair.

The following examples further describe the practice of embodiments of the invention with BMP-2. The examples are not limiting and as will be appreciated by those skilled in the art, may vary according to the above description.

EXAMPLES I. Rabbit Allograft

All the processes were carried out with the approval of IACUC. Twelve white New Zealand rabbits of the 7 males (6 months of age) were used. Two rabbits served as donors and 10 as recipients. Osteocartilaginous grafts (3.5 mm in diameter) were collected from the trochlear pit or medial femoral condyle of the donors and were transplanted into a defect with 3.5 mm depth in the trochlear pit of the recipient. The graft was irrigated with rhBMP-2 (0.5 mg / mL) or with control buffer prior to implantation. Rabbits were sacrificed 4 weeks after surgery and the transplants and surrounding tissue were evaluated through a histological-histochemical classification grid, as described in Sellers et al., J. Bone Joint Surg., 79-A: 1452-1463 (1997). Computed image analysis of histological sections was also performed. The results were evaluated using the unpaired Student t-test.

On superficial examination, the joints showed no sign of inflammation. All defects were filled by the repairing tissue. The surface appearance of the defects was variable but acceptable and did not correlate with the form of treatment. Osteophytes were found in 3 joints (2 in the experimental group, 1 in the control buffer group). There was no correlation between the superficial and histological appearance in any of the defects. The presence of chondrocytes in the gaps and the sporadic cloning of cells in the donor cartilage indicated tissue survival. Focal degeneration of donor cartilage was present in all control groups, but in only one of the rhBMP-2 treated groups. The healing of the defect in the rhBMP-2 treated group was significantly improved compared to that of the control group. The rhBMP-2 treated group had improved bone integration indicated by less fibrous repair tissue in the subcartilagine bone compartment. Treatment with rhBMP-2 also resulted in more cartilage above the original mark, apparently consisting of both donor tissue and newly regenerated receptor cartilage. There was no significant difference in the total amount of bone observed between the two groups.

TABLE I HISTOLOGICAL CLASSIFICATION AND HISTOMORPHOMETRIC MEASUREMENT FOR CARTILAGE REPAIR, MEDIUM VALUE (SD) Parameter rhBMP-2 Control Mean Classification ** 10.0 (5.42) * 20.6 (5.18)% bone under the mark 73 , 26 (13.28) 62.88 (18.07)% of fibrous tissue under the mark 2.19 (2.04) 15.81 (9.88)% cartilage above the mark 74.70 (41, 08) * 18, 17 (26, 70) defect fill% 96.53 (4.86) * 88.79 (8.04) * Statistically control difference (p <0.05). significant relative to the ** scale system ranges from 0 (normal cartilage) to 31 (no repair).

Additional histomorphometric analysis data further support the beneficial effects of rhBMP-2 on graft healing. For example, the percentage fill of the new tissue above the mark was found to be 81.52% in a group treated with rhBMP-2 vs. 57, 63% in the control. There was less degeneration of graft cartilage in the group treated with 9 rhBMP-2 (23.83%) than in the control group (44.52%). Integration of newly formed graft or cartilage with host cartilage was improved by treatment with rhBMP-2 (56.48%) as compared to that of the control (21.89%) group. Further new cartilage was formed under the influence of rhBMP-2 either at the edge of the graft, where it eliminated the cleft between the graft and the host either at the top of the graft, where it made the graft more congruent with the surface of the joint.

The results above demonstrate that the healing of allogeneic osteocartilaginous grafts in articular cartilage defects was improved by the addition of rhBMP-2. Active growth factor may have accelerated the subcartilaginous bone union, providing support and nutrition to articular cartilage tissue. The addition of growth factor may also have stimulated the formation of new cartilage from mesenchymal receptor stem cells in bone marrow and / or synovial tissue. These results suggest that the combination with active growth factor, particularly bone morphogenetic proteins and osteocartilaginous allografts may offer a potent strategy for the treatment of articular cartilage defects, particularly total joint cartilage thickness defects. II. Autografting in Rabbit

Osteocartilaginous grafts (2.7 mm in diameter and 3.0 mm in length) were collected from the trochlear pit or the femoral condyle and were transplanted at a donor site 2.7 mm wide and 3.5 mm long at the pit trochlear or in the femoral condyle of the knee joint in rabbits. Half of the animals were buffered dropwise at the receptor site prior to transplantation, and then the grafts were submerged in buffer for 2 minutes and placed in the recipient site. 5æg of rhBMP-2 was given dropwise at the recipient site before transplantation to the other half, then the graft was submerged for 2 minutes in buffer containing rhBMP-2500 pg / ml and then transplanted into the site receptor. The animals were sacrificed 4 weeks after surgery and the recipient sites were histologically evaluated using both a histological-histochemical classification grid [Sellers, et al., J. Bone Joint Surg., 79-A: 1452-63 (1997) ] as quantitative computerized image analysis of the tissue. The data indicated that treatment with rhBMP-2 improved autograft healing. The most significant effects were the reduction of graft cartilage degeneration (8.18% for rhBMP-2 vs. 36.25% for control) and more cartilage formed at the border of the graft (88.23% for rhBMP -2 vs. 50% for the control). III. Autograft in Nonhuman Primate:

The non-human primates used for autograft experiments were cynomologous monkeys. Osteocartilaginous grafts (3.5 mm in diameter x 6 mm in length) were collected from the trochlear fossa of 6 cynomologous monkeys and were transplanted at recipient sites punctured at the femoral condyle both medial and lateral of the same animal (n = 12 total transplants ). Prior to transplantation, 25 μg of rhBMP-2 were administered dropwise at 6 receptor sites and the grafts of these transplants were submerged in a 1.25 mg / ml rhBMP-2 solution for 2 minutes. In the other 11 6 transplants, only buffer was administered dropwise at the recipient sites and the grafts were submerged in buffer only for 2 minutes prior to transplantation. The limbs were immobilized in a mold for 2 weeks after the operation and the animals were sacrificed 9 weeks after the operation.

All animals had the normal function of their knee joints. On superficial examination, the joints showed no sign of inflammation. No osteophytes were found at any joint. Although the surface of the defects appeared to level with the surrounding cartilage on the superficial examination, microscopic observation revealed a decrease of the grafts in most cases. The tissue observed by superficially coating the surface was, in fact, tissue formed again at the top of the graft. Computed image analysis was performed by a randomized assessor to quantify defect fill percentage, new tissue types formed above the original mark and integration of the grafts with the surrounding cartilage. Favorable results were observed in the rhBMP-2 treated group at all of these parameters. Further new cartilage was formed between the graft and host cartilage to eliminate the gap resulting in better integration of the graft with the surrounding cartilage (88.59% for rhBMP-2 vs. 64.82% for control). The cartilage defect filling was better in the rhBMP-2 treated group (95.02%) than in the control group (86.68%). There was more fibrous tissue in the control group (11.90% vs. 5.65% for rhBMP-2), while more transitional tissue was found in the rhBMP-2 treated group (36.38% vs. 20.53 % for the control). There was no significant difference in the overall histological-histochemical classification between the two groups. Peripheral computerized quantitative tomography (pQCT) showed that bone density increased over time at donor sites. At 6 weeks and 9 weeks post-operatively, the tissue at the rhBMP-2 treated donor sites was significantly dense and the healing process was more advanced compared to the control sites. Histologically, donor sites contained regenerated bone trabeculae with fibrous tissue on the surface in all cases. IV. Retention of rhBMP-2 Ex Vivo:

Retention of rhBMP-2 in the osteocartilaginous graft with this technique was evaluated in non-human primates grafts. The graft was submerged in a 125 I-labeled rhBMP-2 mixture solution and unlabeled rhBMP-2. The results showed that the amount of rhBMP-2 absorbed by the graft was proportional to the concentration of the protein and the time of infiltration. Other factors affecting the retention of rhBMP-2 included graft size and the presence of bone marrow elements between the trabecular bone. V. Retention of rhBMP-2 over Time In Vivo:

Retention of rhBMP-2 over time in the osteocartilaginous graft was evaluated in rabbits. An unlabelled 125 I and rhBMP-2 blending solution was applied, which contained 5æg of rhBMP-2 and 20æCi of I, to the graft prior to implantation. The animals were screened with a γ-chamber during the follow-up period for 22 days after the operation. Compared with the evolution over time of the collagen sponge as a carrier, the half time of rhBMP-2 in the osteocartilaginous graft was increased from 1 day to 3 days. The radioactivity of 10% of the initial point was maintained from the 11 days of the collagen sponge until the 22 days of the graft. SAW. Non-human primate allografts:

Donor sites (3.5 mm wide x 6 mm long) were removed from the trochlear pits of 12 adult cynomologous monkeys and transplanted at 3.5 x 6 mm receptor sites into the medial and lateral femoral condyles of unrelated individuals. Half of the transplants were soaked in 1.25 mg / mL rhBMP-2 for 2 minutes before transplantation and half was soaked in buffer. An identical process was performed on the other member 7 weeks after the first surgery. The limb was immobilized in a template for 2 weeks after the operation after each surgery and the animals were sacrificed 9 weeks after the second surgery for histological analysis.

These results suggest that the combination between active growth factor, particularly bone morphogenetic proteins and osteocartilaginous autografts may offer a potent strategy for the treatment of articular cartilage defects, particularly total joint defects of the articular cartilage. In other embodiments BMP-2 may also be applied to frozen osteocartilaginous allograft for the treatment of focal cartilage defect of the articular cartilage.

The foregoing descriptions detail presently preferred embodiments of the present invention. It is expected that numerous modifications and modifications to the practice will occur to those skilled in the art upon appreciation of these disclosures. Such modifications and variations are contemplated to be within the scope of the claims appended hereto.

Lisbon, February 3, 2010 15

Claims (10)

The use of at least one purified bone morphogenetic protein (BMP) in the preparation of a medicament for use in a method for improving the integration of an osteocartilaginous graft, comprising the use of administering BMP to a region in need of regeneration of the cartilage prior to administration of the osteocartilaginous graft, having applied thereto an amount of BMP that is chondrogenic and effective in stimulating the activity of infiltrating progenitor cells. The use of claim 1, wherein BMP is BMP-2. The use of claim 1 or 2, further comprising administering a protein selected from the group consisting of BMP-12, BMP-13 and MP52. The use of any one of claims 1 to 3, wherein the medicament is intended to improve retention of BMP at the delivery site. The use of any one of claims 1 to 3, wherein the osteocartilaginous graft is an osteocartilaginous autograft obtainable by mosaicoplasty. A composition comprising at least one purified bone morphogenetic protein (BMP) for use in a method for improving the integration of an osteocartilaginous graft, comprising the use of administering BMP to a region in need of regeneration of the articular cartilage prior to administration of the osteocartilaginous graft, having applied thereto an amount of BMP which is chondrogenic and effective in stimulating the activity of infiltrating progenitor cells. The composition of claim 6, wherein BMP is bmp-2. The composition of claim 6 or 7, further comprising administering a protein selected from the group consisting of ΒΜΡ-12, ΒΜΡ-13 and ΜΡ52. The composition of any one of claims 6 to 8, wherein the composition is intended to improve retention of BMP at the delivery site. The composition of any one of claims 6 to 8, wherein the osteocartilaginous graft is an osteocartilaginous autograft obtainable by mosaicoplasty. Lisbon, February 3, 2010 2